Antioxidant, anti-amylase, anti-lipase, and efficiency of Satureja fatty acid on the anti-inflammatory parameters in lipopolysaccharide-stimulated macrophage through Nrf2/NF-kB/NADH oxidase pathway

Satureja is an aromatic plant that is used for flavoring, perfume, and food manufacturing due to its pleasant essential oil. Modern medicine research revealed several biological activities of Satureja essential oil, including antifungal, antibacterial, antiviral, antioxidant, anticancer, and anti-inflammatory. However, the functional properties of Satureja fatty acid have not been explored. This study examined the fatty acid profile, lipid nutritional quality, antioxidant, anti-amylase, and anti-lipase capacities of Satureja. The efficiency of Satureja fatty acid on the anti-oxidative and anti-inflammatory parameters in LPS-induced macrophage through the Nrf2/NF-kB/NADH oxidase pathway was examined. The whole lipid extract was prepared with chloroform/methanol/water solution. Fatty acids methyl ester from whole lipid extract were prepared with methanol/sulfuric acid reagent. The fatty acid profile was analyzed using gas chromatography-mass spectrometry. Total antioxidant was determined by ABTS decolorization. Lipase and amylase activities were determined by monitoring the decomposition of p-nitrophenyl butyrate and starch. The macrophage cell line was grown in DMEM media in the presence of fatty acid. The hydrogen peroxide production in treated cells was monitored using the FOX reagent. NADH oxidase activity was measured by monitoring NADH breakdown. The expression of NOX, NF-kB, and NRF2, were tested in the treated cells by real-time PCR. The main components of the Satureja fatty acid were linolenic acid (24.67–37.32%), palmitic acid (10.65–20.29%), linoleic acid (8.31–13.39%), oleic acid (4.42–14.35%), stearic acid (2.76–8.77%) and palmitoleic acid (1.77–4.95%). Given the nutritional quality, omega-3 PUFA (23.58–37.32%), SFA (21.53–26.70%), omega-6 PUFA (10.86–16.14%), omega-9 MUFA (4.42–14.35%), and omega-7 MUFA (1.77–4.95%) comprise the majority of fatty acids. Satureja fatty acid has a promising unsaturation index (120.77–164.27), PUFA/MUFA (2.07–6.41), hypocholesterolemic index (2.44–3.47), health-promoting index (2.03–2.42), PUFA/SFA (1.37–1.94), nutritive value index (0.53–1.71), MUFA/SFA (0.30–0.80) omega-6/omega-3 (0.34–0.65), atherogenicity index (0.41–0.49), and thrombogenicity index (0.17–0.27). Satureja fatty acid displayed strong antioxidant capacity (with IC50 ranging from 354 to 428 µg/mL), anti-lipase capacity (with IC50 ranging from 354 to 428 µg/mL), and anti-amylase capacity (with IC50 ranging from 370 to 390 µg/mL). LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells. In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and increased NRF2 at 0.04 mg/mL. In conclusion, Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities. The mechanisms in lowering oxidative stress markers depended on down-regulating superoxide-producing enzymes at gene and protein levels. Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.


Herbal materials
Satureja hortensis L. leaves were taken from an experimentation field.Following identifying the plant species, the voucher specimens (Herbarium number 538F19) were added to the herbarium at Shiraz University.Satureja bakhtiarica Bunge (Herbarium number MPH-1577) was collected from Semirom, Esfahan (central part of Iran).Aerial parts of Satureja sahendica Bornm (Herbarium number: MPH-1582) and Satureja khuzistanica Jamzad (Herbarium number MPH-1588) from Mazhin, Lorestan, at the whole flowering stage were collected.Satureja rechingeri Jamzad (Herbarium number: MPH-1348) and Satureja mutika (Herbarium number MPH-1356) from Dehloran, Ilam (western parts of Iran) at the whole flowering stage were collected 13 .Professor Ahmad Reza Khosravi (Faculty of Science, Shiraz University, Shiraz, Iran), an experienced botanical taxonomist, recognized and confirmed this plant taxonomically.The plant material is dried in the shade and ground into powder using a household blender for later use.Plant materials were dried in the shade and powdered by the home grinder.
Experimental research and field studies on cultivated and wild plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation.This plant grows naturally in the different plains of Iran Natural Resources.The plants were collected to comply with relevant national policies and legislation of the Fars Agricultural and Natural Resources Research and Education Center (Fars, Iran).Permission was issued to collect the plant materials for this investigation.Agricultural and Natural Resources Research and Education Center confirmed all the source and batch number details.The qualitative chemical components of Satureja spp were considered using Fourier transform infrared (FT-IR) spectroscopy with FTIR spectrophotometer (Bruker, Germany) 14 .

Fatty acids preparation and profiling
A modified and developed method of Folch containing chloroform: methanol: water (2:1:0.8,v/v/v) was used to extract and prepare total lipid 15 .Fatty acids methyl ester from whole lipid extract (1.0 g) were prepared with 5.0 mL of acidic methanol (methanol: sulfuric acid in 80:20 v/v) for 120 min at 80 °C.At that time, 5.0 mL of NaCl (0.7%) was added and vortexed for 5.0 min.Then, 5.0 mL of hexane was added and vortexed for 5.0 min, then centrifuged at 3000×g for 10 min.The fatty acid profile was analyzed using an Agilent gas chromatograph (Agilent 7890B GC 7955A MSD) coupled with a single quadrupole mass spectrometer and equipped with a fused silica capillary HP-5MS column (30 m × 0.25 mm; thickness 0.25 µm) according to previously published work 14 .The fatty acid was diluted in hexane, and 1.0 µL was injected into the column.The initial and maximum temperatures were 50 °C and 300 °C, respectively.The electron impact ionization mode was used for the mass spectrometric detection.Mass spectral was obtained from 40 to 650 Da at 70 eV electron ionization energy.The scanning time was 58 min.The split ratio was 1:50.The source and quadrupole were adjusted to 230 °C and 150 °C, respectively, while the interface was heated to 240 °C.The Oven temperature program was 60-280 °C as follows: 60 °C for 1.0 min, rise at 5 °C/min to 220 °C, and rise at three °C/min to 280°C 14 .The fatty acid components were determined by comparing the related peak retention times with those reported in the libraries (Wily 7n and NIST05a).

Total antioxidant capacity of Satureja fatty acid
The total antioxidant capacity was evaluated by mixing the Satureja fatty acid (0.02 mL equal to 20 mg) with 1.0 mL of ABTS radical solution (2.54 mM potassium persulfate plus seven mM ABTS) and monitoring light absorbance at 734 nm.The percentage of radical inhibition and the 50% inhibitory concentration (IC 50 ) were calculated using the change in absorbance at 734 nm.A calibration graph was drawn based on Trolox (1.0 mg/ mL) as a standard reference.The antioxidant potential of Satureja fatty acid was measured in milligrams of Trolox equivalents (TE) per gram 17 .All measurements were performed in triplicate.

Lipase inhibition
The fatty acid was dissolved at 20 mg per mL (equal to 0.02 mL/mL) in tween-80 (0.3%).To determine the lipase (EC 3.1.1.3,from Saccharomyces cerevisiae, Sigma-Aldrich, St Louis, MO, USA) inhibitory activity, lipase (2.0 U/mL) was incubated with Satureja fatty acid and orlistat (0.03 mL, 0.03-0.6mg/mL) for 30 min at 37 °C.p-nitrophenyl butyrate (pNPB, 0.03 mL, 10 mg/mL) was added to the mixture to start the reaction.For 30 min, light absorption at 405 nm was measured.The milligrams of orlistat equivalents per gram of samples were used to measure the anti-lipase activity.Using the change in absorbance, the inhibitory concentration that inhibits lipase by 50% (IC 50 ) and the lipase inhibition percentage was computed 18 .In the presence of inhibitors, the Lineweaver-Burk plot was drawn based on 1/[pNPB] versus 1/velocity to study the lipase kinetics.The substrate and inhibitors were pNPB (0.0-10 mg/mL) and orlistat (0.3 mg/mL) or Satureja fatty acid (0.3 mg/mL), respectively.The quantitative pNPB breakdown per minute was determined using light absorbance monitoring at 405 nm.The lipase activity was shown by the slope of changes in light absorbance over time.The kinetic parameters of lipase inhibition were determined through the Lineweaver-Burk plot.

Amylase inhibition
To determine the α-amylase (EC 3.2.1.1,from Bacillus sp., Sigma-Aldrich, St Louis, MO, USA) inhibitory activity of Satureja fatty acid, the solution of amylase (0.04 mL of 2.0 U/mL) was pre-incubated with inhibitors (0.04 mL of 0.03-0.60mg/mL of acarbose or Satureja fatty acid) at 37 °C for 20 min in a 5.0 mL vials.After the incubation, starch solution (0.25 mL of 10 mg/mL) was added.The mixture was incubated at 37 °C for 30 min.In the end, 0.10 mL of iodine reagent was added, and light absorbance was recorded at 580 nm 14 .The anti-amylase activity was expressed in milligrams of acarbose equivalents per gram of samples using the calibration graph with the positive control (acarbose).The inhibitory concentration resulting in 50% amylase inhibition (IC 50 ) was calculated based on the change in light absorbance 19 .In the presence of Satureja fatty acid (0.30 mg/mL), the Lineweaver-Burk plot was employed for amylase kinetic assay based on the 1/[starch] versus 1/velocity plot.The starch (0.0-10 mg/mL) was used as substrate.Acarbose (0.30 mg/mL) and Satureja fatty acid (0.30 mg/mL) were used as inhibitors.The rate of starch breakdown for 30 min was measured using light absorbance monitoring at 580 nm.Amylase activity is determined by the slope of light absorbance change (starch degradation) versus time.The kinetic parameters of amylase were determined using the Lineweaver-Burk plot 12 .

Fluorescence and ultraviolet spectroscopy analysis
Using ultraviolet and fluorescence spectroscopy, the interactions of amylase and lipase with Satureja fatty acid were examined 20 .Briefly, 0.5 mL of inhibitor solutions were added to the 0.5 mL of amylase or lipase solutions (5.0 mg/mL).The mixtures were placed for 10 min at ambient temperature.The UV absorption spectra of the enzyme-inhibitor solution were recorded using an ultraviolet absorption spectrophotometer (UV1280, Shimadzu, Japan).A fluorescence spectrophotometer (Varian Cary Eclipse, Agilent, USA) was used to study the intrinsic fluorescence absorption of enzyme-inhibitor solutions at excitation wavelengths of 280 nm and emission wavelengths of 290 to 500 nm.

Macrophage cell line culture
The hematopoietic mouse macrophage cell line (J774.A1, NCBI code, C483) was purchased from the cell bank of the Pasteur Institute of Iran.The macrophage cell line was grown in DMEM media supplemented with FBS (fetal bovine serum, 10%), glutamine (2.0 mM), streptomycin (0.10 mg/mL), and penicillin (100 U/mL).Cytotoxic effects of Satureja fatty acid on the macrophages were assessed using an MTT assay.Macrophage cells and Satureja fatty acid were added to tissue culture plates at concentrations ranging from 0.0 to 0.2 mg/mL.The supernatant was then mixed with the MTT solution (0.5 mg/mL), and the mixture was incubated for four hours at 37 °C.The MTT was replaced with DMSO to dissolve the formazan crystals.Light absorption at 492 nm was measured to evaluate cell viability.Macrophage cells were incubated with LPS (2.0 mg/mL) and non-cytotoxic concentrations of Satureja fatty acid to assess their inflammatory marker.The expression of NOX, NF-kB, NRF2, and hydrogen peroxide production were tested in the treated cells 21 .

Hydrogen peroxide measurement
The hydrogen peroxide production was monitored using FOX (Ferrous oxidation-xylenol orange) reagent.The FOX solution contained xylenol orange (0.250 mM), ferrous ions (0.250 mM), and perchloric acid (110 mM).The culture medium (0.9 mL) and methanol (0.1 mL) were mixed, and the combination was incubated at room temperature for 30 min.After adding the FOX reagent (0.9 mL), the mixture was incubated for 30 min, and light absorbance at 560 nm was measured 22 .

NOX activity
Sodium dodecyl sulfate (SDS) is used to lyse macrophage cells.The NOX activity is measured using a reaction solution containing dithiothreitol (1.0 mM), sodium phosphate (100 mM), and NADH (100 mM).The reaction began when 0.5 mL of the reaction solution was added to 0.5 mL of the cell extract.NADH breakdown is followed by the measurement of light absorbance at 345 nm.The decomposition of 0.001 mM NADH per minute is the unit of NOX activity 23 .

Whole RNA obtaining and analysis of the real-time PCR
Total RNA is obtained using an RNA extraction kit (TRNX-plus, Cinagen, Tehran, Iran).First-strand DNA reagent (Fermentas, Hanover, MD) was used to synthesize first-strand cDNA from 0.001 mg of mRNA.A secondstrand cDNA kit (Invitrogen, ThermoFisher Scientific) was used to synthesize second-strand cDNA using specific primers (Table S2 in supplementary file).The thermal program and the amplification cycles performed in the thermal cycler were completed (Line-Gene, Bioer Technology Co., Hangzhou, China).The threshold cycle (CT) was used to estimate the relative expression of the target genes.The Line-gene K program was used to compute the CT value for each gene 24 .

Statistical evaluation
The data are presented as mean values plus standard deviations and derived from at least three research studies.One-way analysis of variance (ANOVA) and Tukey post-hoc tests were used to examine significant differences between treatments using the statistical package for the social sciences (SPSS, Abaus Concepts, Berkeley, CA) software.Relationships between the different samples and the association between essential oil profile and antioxidant, anti-amylase, and anti-lipase capacities were analyzed by principal component analysis (PCA).Minitab 18 statistical software was used to perform the PCA based on the correlation matrix.www.nature.com/scientificreports/

FTIR spectroscopy
According to FT-IR analyses, Satureja had a high carbohydrate content, moderate protein and fatty acids, and low phenolic monoterpenes and monoterpenes (Fig. 1).The broad peak observed at 3800-3100 cm −1 indicated the presence of the OH functional group due to the stretching vibration of water or hydrogen in hydrogen bonds.Such peaks stated the existence of alcohol and phenyl groups.The bands at 2930-2900 cm −1 can be attributed to C-H vibrations, including CH, CH 2 , and CH 3 stretching of fatty acids, hydrocarbon chain of amino acid, aliphatic chain, alkynes, and aldehydes.The peaks at 1750-1500 cm −1 were consigned to the C=O stretch, the C=C stretch of aromatic compounds, amide I and II, and amide II of the protein component.Stretching of carbohydrates C-O, C-C, and C-O-H are reflected in the peaks at 1150-100 cm −1 .The bands at 1100-900 cm −1 resulted from C-O-H and =CH bending of carbohydrates.The 1000-800 cm −1 spectrum band can be assigned to C-H bending due to terpenes 25 .The Satureja fatty acid had a high level of fatty acid, especially unsaturated types, and low levels of monoterpenoids and monoterpenes (Fig. 2).
To treat and prevent hypertension, inflammation, obesity, anti-diabetes, and metabolic syndrome, Satureja fatty acid could be employed as a food ingredient and dietary supplement 28 .

Total antioxidant activity
Satureja fatty acid displayed antioxidant capacity with IC 50 ranging from 354 to 428 µg/mL.Satureja fatty acid showed antioxidant capacity ranging from 410 to 475 mg Trolox/g.These values were equivalent to 37.27 to 43.18 mg Trolox equivalent per gram of Satureja dried powder (Table 2).These experimental results suggested that the Satureja fatty acid, which contains monoterpenoids (thymol and carvacrol) and polyunsaturated omega-3 and omega-6 fatty acids, display potent antioxidant activity.To some extent, these experimental results are similar to those of Oliveria decumbens, Thymus kotschyanus, Trachyspermum ammi, and Zataria multiflora fatty acids 19 .
A growing body of evidence supports the therapeutic potential of omega-3 polyunsaturated fatty acids (n-3 PUFA), mainly docosahexaenoic (DHA) and eicosapentaenoic acid (EPA), on metabolic diseases based on their antioxidant and anti-inflammatory properties 29 .Some studies suggested that long-chain polyunsaturated fatty acids displayed antioxidant activity and thus can scavenge superoxide and reactive radicals in an unsaturationdependent manner 30 .Furthermore, emerging evidence suggests that the antioxidant and anti-inflammatory properties of polyunsaturated omega-3 fatty acids seem to play a vital role in their effectiveness in patients with cardiovascular pathologies 31 .

Anti-lipase activity and kinetic analysis
Triacylglycerols are broken down into fatty acids and glycerol by pancreatic lipase.Pancreatic lipase inhibitors can stop fatty acids from breaking down and entering the blood, which helps to lower lipid levels that can prevent obesity.Anti-obesity capacity was calculated based on lipase activity inhibition assay, and the results were reported as IC 50 and orlistat equivalents per gram (Table 3 and Fig. 3).Satureja fatty acid displayed anti-lipase capacity with IC 50 ranging from 270 to 293 µg/mL.Satureja fatty acid showed anti-lipase capacity ranging from 824 to 887 mg orlistat equivalent per gram oil.These values equal 75 to 80.6 mg orlistat per gram of Satureja dried powder (Table 3 and Fig. 3).Fatty acids are competitive inhibitors of lipases.Lipases hydrolyze ester bonds between fatty acids and glycerol; free fatty acids did not have an ester bond for hydrolysis.Fatty acids with a similar structure to monoacylglycerol can bind to the active site of lipase without any hydrolysis reaction and inhibit lipase 32 .Monoterpenes, monoterpenoids, omega-3/omega-6 fatty acids, or the combined effects of these compounds can all be associated with this lipase inhibitory capacity 33 .
The lipase K m /V max increased while K m and V max declined in the presence of the Satureja fatty acid, according to the kinetic research based on the double reciprocal plot (Table 3 and Fig. 3).Satureja fatty acid inhibits lipase through competitive and non-competitive techniques.Monoterpenes, monoterpenoids, omega-3, and omega-6 fatty acids may all have a role in lipase inhibition.By raising the enzyme K m and limiting triglyceride breakdown, small molecules can either occupy the triglyceride-binding site of lipase or act as triglyceride blockers.Large molecules can bind to allosteric regions in lipase, causing a conformational change, lowering lipase affinity for triglyceride, lowering Vmax, and slowing triglyceride breakdown.Conformational changes and activity inhibition occur when these bioactive compounds bind to active and allosteric sites 34 .

Anti-amylase and kinetic analysis
Diabetes is characterized by hyperglycemia and glucose digestion system disturbances caused by an insulin section or action deficiency.In this case, amylase is an important enzyme that converts starch and oligosaccharide to glucose and affects blood glucose levels.Anti-diabetic activities were calculated based on amylase activity inhibition assay, and the results were reported as IC 50 and acarbose equivalents per gram.Satureja fatty acid displayed anti-amylase capacity with IC 50 ranging from 370 to 390 µg/mL.Satureja fatty acid showed anti-amylase capacity  4 and Fig. 3).These experimental results are similar to Oliveria decumbens, Thymus kotschyanus, Trachyspermum ammi, and Zataria multiflora fatty acids 19 .There is rare evidence that fatty acids, lipids, saponins, and lipophilic compounds found in fruits, vegetables, and mushrooms contribute to the in vitro α-glucosidase and α-amylase inhibition activities 35,36 .However, limited work has been conducted on the elucidation of the α-glucosidase and α-amylase inhibition activities of different fatty acids and lipids.Future studies are encouraged to investigate the individual inhibitors of cereal lipids and their inhibition mechanism.However, these rare results revealed that the presence of double bonds may play a crucial role in the anti-amylase activity of fatty acids.Additionally, unsaturated (oleic acid, linoleic acid, linolenic acid) and saturated (palmitic acid, stearic acid) fatty acids have been reported to have glucosidase and amylase inhibitory effects as competitive/uncompetitive inhibitors of starch-digestive enzymes, despite not structurally resembling carbohydrates 35,36 .
The double reciprocal plot suggests that in the presence of the fatty acid from Satureja, the amylase K m /V max increased, and V max decreased (p < 0.05).Satureja fatty acid did not have significant effects on the K m of amylase.

Ultraviolet and fluorescence spectroscopic analysis
Ultraviolet-visible light absorption and fluorescence quenching analysis effectively reveal enzyme-inhibitor interactions.The ultraviolet spectrum of amylase (Fig. 4) and lipase (Fig. 4) at 270-280 nm increased in the presence of fatty acid.The fluorescence emission of amylase (Fig. 4) and lipase (Fig. 4) is reduced in the presence of the fatty acid.As a result, Satureja components can form non-covalent complexes with amylase and lipase, modify their conformation, and depict the aromatic group to Ultraviolet.Amylase and lipase ultraviolet light absorption increases, and fluorescence quenching establishes the non-covalent interaction between amylase and lipase and fatty acid components and reflects conformation changes 37 .These interactions depict the aromatic group to ultraviolet light leading to light absorption, on the other hand, producing a non-fluorescent enzyme-inhibitor compound, altering the enzyme microenvironment and architecture, and lowering the intrinsic fluorescence intensity 20 .

Cytotoxicity of Satureja fatty acid
The fatty acids from Satureja reduced the viability of macrophage cells in a concentration-dependent manner.
The cytotoxicity rapidly increased at doses greater than 0.12 mg/mL (Fig. 5).Fatty acids are lipophilic substances that enter cells, disrupt organelle membranes, and cause cytotoxicity by penetrating and disrupting the cytoplasmic membrane.The fatty acid has a negligible effect on cytoplasm membranes and mitochondria at low concentrations.The presence of monoterpenes in the Satureja fatty acid can also destroy protein, DNA, and RNA, impair mitochondria, and cause cell death 38 .The cytotoxic effects of lipophilic compounds are ascribable to the lipophilic nature of the constituents that allow them to cross cell membranes, altering the phospholipid layers, increasing membrane fluidity, and leading to ions and cytoplasmic content leakage.The alteration of pH gradient reduced ATP production, and loss of mitochondrial potential are just a few consequences of disturbed cellular membranes 39 .www.nature.com/scientificreports/

Expression of oxidative biomarkers in cells treated with LPS
The levels of hydrogen peroxide, NOX, NF-kB, and NRF2 were low in untreated cells.Following LPS treatment, hydrogen peroxide, NOX, NF-kB, and NRF2 levels significantly rise (Table 5 and Figs.S1 and S2 in supplementary file).When LPS-stimulated cells were exposed to the non-toxic (0.04 mg/mL) concentration of the fatty acid of Satureja, the levels of hydrogen peroxide, NOX, and NF-kB activity were considerably decreased.At the same time, NRF was increased (Table 5 and Figs.S1 and S2 in supplementary file).These experimental results, to some extent, are comparable with the results of Najafi et al. 21on Agastache foeniculum fatty acids, Aminzadeh et al. 40 on the Zataria multiflora essential oil, and the results of Obeidnejad et al. 22 on Satureja bakhtiarica essential oil.It has been demonstrated that LPS causes superoxide and hydrogen peroxide synthesis and oxidative damage.Hydrogen peroxide and superoxide anion are mitogen-activated protein kinases (MAPK) stimulants.MAPKs induce NF-kB to increase the production of cytokines and inflammatory mediators.When LPS binds to the toll-like receptor 4 (TLR4), it triggers the translocation of NF-kB into the nuclei, which causes the production of several pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 41 .Furthermore, it has been discovered that consuming extra omega-3 in the diet provides anti-inflammatory effects against several inflammatory disorders, including osteoarthritis and inflammatory bowel disease 42 .By inhibiting the TLR4/NF-kB signaling pathway, omega-3 can lessen the release of pulmonary inflammatory factors and oxidative stress in rats following acute lung damage 43 .Contrarily, omega-6 fatty acids are the building blocks for some pro-inflammatory mediators, such as prostaglandins and leukotrienes.Long-chain omega-6 fatty acids tend to produce pro-inflammatory eicosanoids, whereas omega-3 promotes the development of antiinflammatory eicosanoids 44 .These results suggested that Satureja fatty acids with high omega-3 fatty acids can diminish hydrogen peroxide production and have anti-oxidative solid capabilities in stimulated macrophages.
Oxidative stimuli like LPS activate NRF2 and NF-kB.The NF-kB-IKB complex is the inactive variant of NF-kB in unstimulated cells.Inflammatory response and oxidative product are strengthened due to IKB release and NF-kB activation.Typically, the inactive form of NRF2 is the NRF2-Keap1 complex.In oxidative stress, Keap1  www.nature.com/scientificreports/ is released from NRF2 and activates NRF2.The NRF2 activation improves antioxidant defenses and superoxide neutralization coming on by NF-kB pathway activation.NRF2 is a key transcription factor that controls many aspects of cell homeostasis in response to oxidative stress.The target genes of NRF2 encode the antioxidant proteins, metabolic enzymes, transporters, redox balancing factors, and glutathione-conjugated coenzyme 45 .
The NF-κB family regulates many genes involved in cellular processes, such as cell differentiation, proliferation, development, and apoptosis.The NRF2 and NF-κB pathways co-regulate cellular responses to oxidative stress and inflammation.In the cross-talk mechanism between NRF2 and NF-κB pathways, NRF2 and NF-κB compete for translocation to the nucleus, which depends on the relative amount of NRF2 and NF-κB.Furthermore, NRF2 is indirectly activated by anti-inflammatory compounds that suppress NF-κB activity; likewise, NF-κB is indirectly started by NRF2 inhibitors 46 .Satureja fatty acid had different modulatory effects on NF-kB and NRF2 because they significantly lowered NF-kB expression while raising NRF2 expression in the treated cells.These results point to their potential application as a preventive and therapeutic agent for oxidative damage brought on by inflammation.

Conclusion
These findings showed that the main ingredients in Satureja fatty acids were linolenic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, and palmitoleic acid.Omega-3 PUFA, SFA, omega-6 PUFA, omega-9 MUFA, and omega-7 MUFA comprise the majority of fatty acids from Satureja.Satureja fatty acid has a promising unsaturation index, hypocholesterolemic index, health-promoting index, nutritive value index, omega-6/omega-3, atherogenicity index, and thrombogenicity index.There is no report on the nutritional quality of Satureja fatty acid.However, after reviewing the literature and comparing the nutritional quality of fatty acids with other vegetable oils, we found that the nutritional quality of Satureja is healthier than that of common vegetable oils and is similar to that of meat and dairy products.Satureja fatty acid displayed strong antioxidant capacity, anti-lipase capacity, and anti-amylase capacity.LPS induced the expression of NOX, NRF2, and NF-kB and the synthesis of hydrogen peroxide in macrophage cells.In LPS-stimulated macrophages, Satureja fatty acid reduced NOX expression, hydrogen peroxide, and NF-kB expression and elevated NRF2.Satureja fatty acids have potent antioxidant, anti-amylase, anti-lipase, and anti-inflammatory activities.The common aspect between diabetes, obesity, and inflammation is oxidative stress.The mechanisms in lowering oxidative stress markers depended on www.nature.com/scientificreports/down-regulating superoxide-producing enzymes at gene and protein levels.Satureja polyunsaturated omega-3 fatty acids could be recommended for healthy products combined with dietary therapy to treat obesity, diabetes, and oxidative stress.However, there is very rare evidence of fatty acids composition and quality and their functional activity on the in vitro and in vivo antioxidant and anti-inflammatory activities, as well as carbohydratedigesting and lipid-digesting enzymes.Future studies are encouraged to investigate the molecular mechanism of oxidative stress and inflammatory response mitigation by omega-3 PUFA.

Figure 3 .
Figure 3. Amylase (A) and lipase (C) inhibition activity and Lineweaver-Burk plots of lipase (B) and amylase (D) inhibition in the presence of essential oil from S. bakhtiarica.

Figure 4 .
Figure 4. Ultraviolet absorbance (A,C) and Fluorescence intensity (B,D) of lipase (A,B) and amylase (C,D) in the presence of essential oil from S. bakhtiarica.

Figure 5 .
Figure 5. Cytotoxic activity of Satureja fatty acid on the viability of macrophages cell line.

Table 1 .
Fatty acid and monoterpenes composition (%) of Satureja.The values are expressed as means ± SD for three replicates (n = 3).Mean values with different letters within a row are significantly different (p < 0.05).

Table 2 .
Total antioxidant activity of Satureja lipid hydrolysate.Antioxidant capacity was expressed as the concentration needed for 50% (IC 50 ) radical scavenging and Trolox equivalent (TE).The values are expressed as means ± SD for three replicate experiments (n = 3).The mean values with different letters within a column are significantly different (p < 0.05).

Table 3 .
Anti-lipase capacity (IC 50 ), orlistat equivalent (OE), and kinetic parameters (Km/Vmax, Km, and Vmax) of lipase in response to orlistat and fatty acid from Satureja.The values are expressed as means ± SD for three replicates (n = 3).Mean values with different letters within a column are significantly different (p < 0.05).

Table 4 .
Anti-amylase capacity (IC 50 ), acarbose equivalent (AE), and the kinetic parameters (Km/Vmax, Km, and Vmax) of amylase in response to fatty acid Satureja.The values are expressed as means ± SD for three replicates (n = 3).Mean values with different letters within a column are significantly different.

Table 5 .
Modulatory effects of oil fraction (40 µg/mL) from Satureja on ROS production, NADH oxidase (NOX) activity, and related mRNA expression in LPS-stimulated macrophages.The values are expressed as means for three replicate experiments (n = 3).Mean values with different letters within a row are significantly different (p < 0.05).